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Quantitative Evaluation of Drug Resistance Profile of Cells Expressing Wild-Type Or Genetic Polymorphic Variants of the Human ABC Transporter ABCC4

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Quantitative Evaluation of Drug Resistance Profile of Cells Expressing Wild-Type Or Genetic Polymorphic Variants of the Human ABC Transporter ABCC4 Article Quantitative Evaluation of Drug Resistance Profile of Cells Expressing Wild-Type or Genetic Polymorphic Variants of the Human ABC Transporter ABCC4 Megumi Tsukamoto 1, Shiori Sato 2, Kazuhiro Satake 1, Mizuki Miyake 1 and Hiroshi Nakagawa 2,* 1 Department of Applied Biological Chemistry, Graduate School of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai 487-8501, Japan; [email protected] (M.T.); [email protected] (K.S.); [email protected] (M.M.) 2 Department of Applied Biological Chemistry, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-568-51-9606; Fax: +81-568-52-6594 Received: 14 April 2017; Accepted: 26 June 2017; Published: 4 July 2017 Abstract: Broad-spectrum resistance in cancer cells is often caused by the overexpression of ABC transporters; which varies across individuals because of genetic single-nucleotide polymorphisms (SNPs). In the present study; we focused on human ABCC4 and established cells expressing the wild-type (WT) or SNP variants of human ABCC4 using the Flp-In™ system (Invitrogen, Life Technologies Corp, Carlsbad, CA, USA) based on Flp recombinase-mediated transfection to quantitatively evaluate the effects of nonsynonymous SNPs on the drug resistance profiles of cells. The mRNA levels of the cells expressing each ABCC4 variant were comparable. 3-(4,5-Dimethyl-2- thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay clearly indicated that the EC50 values of azathioprine against cells expressing ABCC4 (WT) were 1.4–1.7-fold higher than those against cells expressing SNP variants of ABCC4 (M184K; N297S; K304N or E757K). EC50 values of 6- mercaptopurine or 7-Ethyl-10-hydroxy-camptothecin (SN-38) against cells expressing ABCC4 (WT) were also 1.4–2.0- or 1.9-fold higher than those against cells expressing the SNP variants of ABCC4 (K304N or E757K) or (K304N; P403L or E757K); respectively. These results indicate that the effects of nonsynonymous SNPs on the drug resistance profiles of cells expressing ABCC4 can be quantitatively evaluated using the Flp-In™ system. Keywords: ATP-binding cassette (ABC) transporter; ATP-binding cassette subfamily C member 4 (ABCC4); drug resistance; single-nucleotide polymorphism (SNP); multi drug resistance protein 4 (MRP4) 1. Introduction Cancer is one of the chief causes of mortality in many developed countries. The broad-spectrum drug resistance of cancer cells varies across individuals and poses a major challenge to cancer research and treatment [1,2]. Drug resistance of cancer cells and differences in their individual levels are usually caused by the overexpression of ABC transporters and single-nucleotide polymorphisms (SNPs) in their genes, respectively. Thus, SNPs in ABC transporter genes determine the response rate to cancer chemotherapy, and development of easy-to-use and quantitative approaches for the identification of these individual-specific SNPs would help combat cancer cell drug resistance through personalized chemotherapy. The human body expresses 48 ABC transporters, which are further divided into seven sub- families (ABCA–ABCG) based on sequence homology and protein organization [3]. The transporters play critical roles in physiological transport and export of drugs and toxic substances, wherein many endogenous and exogenous substrates are transported across membranes in an ATP-dependent Int. J. Mol. Sci. 2017, 18, 1435; doi:10.3390/ijms18071435 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 1435 2 of 15 manner [4–10]. ABCC4, located on chromosome 13q32.1 encodes for the 1325-amino acid-long human ABCC4 (MRP4), and is widely expressed in various tissues, including the liver, kidney, ovary and blood cells [11–13]. Since the first report in 1999 about the direct link between ABCC4 overexpression and impaired efficacy of nucleoside-based antiviral drugs in a human T-lymphoid cell line [14], ABCC4 has been reported to transport a broad spectrum of xenobiotics, including antiviral, antibiotic, antihypertensive and anticancer drugs such as azathioprine, 6-mercaptopurine, and SN-38 [12–25]. The affinity of ABCC4 for its substrate drugs is altered by some of the ≥140 non-synonymous SNPs in ABCC4 [13,24,25]. The SNP variants of ABCC4 (rs11568658, 559 G > T; rs753414892, 1167 A > G; rs11568668, 1460 A > G; rs3765534, 2269 G > A; rs146708960, 2326 G > A; and rs11568644, 3425 C > T) have been suggested to be associated with reduced function of ABCC4, wherein the cellular disposition of substrates for ABCC4 was altered [13,24–26]. Various quantitative functional analyses of ABCC4 [wild-type (WT) or single-nucleotide polymorphisms (SNPs)] have been performed [13,24,25]. However, thus far, the drug sensitivities of cells expressing WT or SNP variants of ABCC4 have never been quantitatively evaluated, since it is difficult to control the integration number and integration site of the cDNA in the genome using traditional transfection methods for establishing cell lines expressing the exogenous gene. Unlike the traditional system, the Flp-In™ system, which is based on the Flp recombinase-mediated transfection can integrate a single copy of the cDNA into the FRT site generated in the telomeric region of the short arm of one copy of chromosome 12 in Flp-In-293 cells [27]. We have reported that the Flp-In™ system can be used to generate cell lines for quantitatively evaluating the effects of the nonsynonymous SNPs on drug resistance profiles [27–30]. Therefore, in this study, we performed a quantitative evaluation of the drug resistance profiles of the cells expressing the WT or SNP variants (M184K, N297S, K304N, P403L or E757K) of human ABCC4 using the Flp-In™ system. 2. Results 2.1. Levels of ABCC4 mRNA and Protein in Cells Established Using the Flp-In™ System In the present study, we employed Flp-In-293 cells with the Flp-In™ system to establish cells expressing WT or non-synonymous SNP variants of human ABCC4 (Figure 1 and Table 1). Flp-In-293 cells were transfected with the ABCC4 cDNA, which integrated into the FRT-tagged genomic DNA, and were then selected using hygromycin B. The resulting hygromycin B-resistant cells were analyzed using qPCR, where the mRNA levels of ABCC4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured. In the present study, the mRNA levels of ABCC4 were corrected according to those of GAPDH, and the resulting ABCC4 mRNA levels were compared among the established cells to evaluate the success of the Flp-In™ system. Figure 1. Schematic illustration of human ABCC4 and the location of its single-nucleotide polymorphisms (SNPs). Arrows, location of SNPs; ABC, ATP binding cassette (nucleotide binding domain). Int. J. Mol. Sci. 2017, 18, 1435 3 of 15 Table 1. Summary of the non-synonymous SNPs in ABCC4 selected in the present study. Nucleotide Nucleotide Amino Acid Amino Acid Variant rsNumber Position Change Position Change M184K rs45454092 551 t > a 184 Met > Lys N297S rs200387797 890 a > g 297 Asn > Ser K304N rs2274407 912 g > t 304 Lys > Asn P403L rs11568705 1208 c > t 403 Pro > Leu E757K rs3765534 2269 g > a 757 Glu > Lys Data on genetic polymorphic variants of ABCC4 were obtained from the the National Center for Biotechnology Information (NCBI) dbSNP database. As shown in Figure 2, ABCC4 mRNA levels in the cells transfected with ABCC4 cDNA were >42- fold higher than those in Flp-In-293/Mock cells. In contrast, the levels of ABCC4 mRNA were comparable among the cells transfected with ABCC4 cDNA, indicating that the Flp-In™ system functioned in the cells established in the present study. Figure 2. Levels of ABCC4 mRNA in cells established using the Flp-In™ system. The levels of ABCC4 and GAPDH mRNA were measured using qPCR with specific primer sets for ABCC4 and GAPDH, as described in Materials and Methods. Data are calculated as ratios by referring to the GAPDH mRNA levels in the cells and normalized to the ratio of ABCC4/GAPDH. Data are expressed as mean values ± S.D. (n = 5). Statistical analyses for significance were performed using one-way ANOVA and Tukey HSD test (* p < 0.01 compared to the Mock group). Since qPCR clearly showed that the Flp-In™ system functioned in these cells, western blot analysis was performed to evaluate the expression of ABCC4 and GAPDH in these cells, wherein all samples were treated with PNGase F to remove the glycomoieties on ABCC4. As shown in Figure 3, the level of ABCC4 was found to correspond to that of the ABCC4 mRNA, and the level of ABCC4 in the ABCC4 cDNA-transfected cells was much higher than that in Flp-In-293/Mock cells. On the Int. J. Mol. Sci. 2017, 18, 1435 4 of 15 contrary, the levels of ABCC4 in cells expressing ABCC4 (M184K or P403L), ABCC4 (N297S or E757K) and ABCC4 (K304N) were comparable, lower and higher compared to that in cells expressing ABCC4 (WT), respectively. Figure 3. Levels of ABCC4 in cells established using the Flp-In™ system. ABCC4 and GAPDH levels were detected using western blot analysis with specific antibodies for ABCC4 and GAPDH, and their levels were measured using ImageJ (Wayne Rasband, Bethesda, MD, USA) as described in Materials and Methods. ABCC4-specific monoclonal antibody (M4I-10) or GAPDH-specific antibody was used for protein detection in PNGase F-treated cell lysate. The experiments were performed independently more than two times. Data are expressed as mean values ± S.D. (n = 3 or 4). Statistical analyses for significance were performed using one-way ANOVA and Tukey HSD test (* p < 0.05 compared to the Mock group; ** p < 0.05 compared to the WT). Int. J. Mol. Sci. 2017, 18, 1435 5 of 15 2.2. Anticancer Drug Resistance Properties of Cells Established Using the Flp-In™ System We performed the 3-(4,5-Dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to determine and compare the anticancer drug resistance properties of the cells.
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